Summary

Hedgehog and canonical Wnt/β-catenin signaling are implicated in
development of the osteoblast, the bone matrix-secreting cell of the
vertebrate skeleton. We have used genetic approaches to dissect the roles of
these pathways in specification of the osteoblast lineage. Previous studies
indicate that Ihh signaling in the long bones is essential for initial
specification of an osteoblast progenitor to a Runx2+
osteoblast precursor. We show here that this is a transient requirement, as
removal of Hh responsiveness in later Runx2+,
Osx1+ osteoblast precursors does not disrupt the formation of
mature osteoblasts. By contrast, the removal of canonical Wnt signaling by
conditional removal of the β-catenin gene in early osteoblast progenitors
or in Runx2+, Osx1+ osteoblast precursors
results in a similar phenotype: osteoblasts fail to progress to a terminal
osteocalcin+ fate and instead convert to a chondrocyte
fate. By contrast, stabilization of β-catenin signaling in
Runx2+, Osx1+ osteoblast precursors leads to
the premature differentiation of bone matrix secreting osteoblasts. These data
demonstrate that commitment within the osteoblast lineage requires sequential,
stage-specific, Ihh and canonical Wnt/β-catenin signaling to promote
osteogenic, and block chondrogenic, programs of cell fate specification.

INTRODUCTION

The mammalian skeleton forms from three distinct cell lineages
(Nakashima and de Crombrugghe,
2003). Facial bones and the cranium are derived from the neural
crest. The base of the skull, the parietal bones and the axial elements of the
ribs and vertebrae are derived from paraxial mesoderm. The sternum and long
bones are formed from lateral plate mesoderm. Although several cell lineages
contribute to skeletal structures, each gives rise to a common bone
matrix-secreting cell type, the osteoblast. The requirement of this cell type
for the production of bone is clearly demonstrated by the targeted mutation of
Runx2 (Komori et al.,
1997; Ducy et al.,
1997; Otto et al.,
1997) and osterix1 (Osx1; Sp7 - Mouse Genome
Informatics) (Nakashima et al.,
2002), transcriptional regulators expressed in osteoblast
progenitors where both mutants lack all bone.

Bone can be produced by two distinct mechanisms, direct differentiation of
osteoblasts from mesenchymal progenitors (intramembranous ossification, e.g.
skull and face) or by formation of bone on a cartilage scaffold (endochondral
ossification, e.g. remainder of the skeleton). Endochondral ossification, the
principle focus of this study, is initiated by the condensation of multipotent
mesenchymal progenitor cells into structures that anticipate skeletal elements
of the adult (reviewed by Kronenberg,
2003). Chondrocytes are the first cell type to form, starting out
as immature proliferative cells that express type 2 collagen (Col2a1)
that subsequently mature into postmitotic type 10 collagen
(Col10a1)-expressing hypertrophic chondrocytes. Osteoblast
progenitors can first be identified in the inner layer of perichondrial cells
that lie immediately adjacent to the zone of hypertrophic chondrocytes, the
periosteum, where the first bone matrix is deposited. Death of hypertrophic
chondrocytes and vascular invasion result in the formation of a new area of
mineralization, the primary spongiosa, within the shaft of the long bones.

Several lines of evidence implicate Hedgehog (Hh) and canonical Wnt
signaling in the regulation of endochondral ossification (reviewed by
Kronenberg, 2003;
Karsenty, 2003). Indian
hedgehog (Ihh) is expressed by prehypertrophic chondrocytes and plays
an essential role in coordinating the growth and differentiation of
chondrocytes, both directly and through the control of other factors, notably
parathyroid hormone-related peptide (Pthrp; Pthlh - Mouse
Genome Informatics). In addition, Ihh appears to act directly on perichondrial
mesenchyme to initiate an osteogenic program in osteoblast progenitors; in the
absence of an Ihh input, these cells adopt an alternate chondrogenic fate
(Long et al., 2004). The
failure of activation of Runx2, a crucial early determinant of the osteoblast
lineage, indicates that Hh signaling acts to initiate an osteogenic program.
Whether Hh signaling is required at later stages of the osteogenic program has
not been addressed.

Initial in vivo evidence for canonical Wnt activity in osteogenesis came
from studies of human and mouse mutations in low-density lipoprotein
receptor-related protein 5 (Lrp5) which encodes a co-receptor
(together with the frizzled family of multi-pass membrane proteins) for Wnt
ligands. Human genetic analysis identified mutations in LRP5 where
bone mass was increased (activating mutations)
(Boyden et al., 2002), or
decreased, in osteoporosis pseudoglioma syndrome (OPPG; null mutations)
(Gong et al., 2001). These
observations have been supported by parallel mouse studies
(Kato et al., 2002). Together,
these findings pointed to a role for a Wnt-mediated process in regulating bone
mass. More recently, manipulation of the canonical Wnt pathway by regulating
the activity of β-catenin, which together with members of the Lef/Tcf
family forms a transcriptional effector complex for this arm of the Wnt
signaling pathway, has shed light on this mechanism. Canonical Wnt signaling
in osteoblasts appears to regulate the production of osteoprotegerin (OPG), a
factor that acts on the other key cell type of bone metabolism, the
osteoclast, inhibiting osteoclast-mediated bone resorption
(Glass, 2nd et al., 2005).

In addition to Wnt function in the maintenance of bone homeostasis, several
recent developmental studies have suggested that Wnts play a role in the
specification of osteoblasts. Specifically, Hill et al.
(Hill et al., 2005) used a
Prx1-cre line to conditionally inactivate β-catenin function,
where Prx1-cre is active in the developing head and limb mesenchyme
at E9.0 prior to skeletogenesis (Hill et
al., 2005). This resulted in a dramatic reduction in the size of
long bone skeletal elements and in an overall failure to develop bone.
Characterization of osteoblast development in this model demonstrates that
Runx2 expression was detectable; however, these mutants failed to
express Osx1, a Runx2-dependent transcriptional regulator that, like
Runx2, is essential for all bone development. In a second study, Day
et al. (Day et al., 2005) used
Dermo1-cre and Col2α1-cre mouse lines to
remove β-catenin function in mesenchymal condensations prior to
chondrocyte and osteoblast development (a later stage than Prx1-cre),
and in cartilaginous condensates prior to the specification of osteoblasts,
respectively. In both models, an overall reduction in the size of skeletal
elements was observed that was accompanied by an apparent arrest of osteoblast
development at a Runx2+, Osx1+ precursor stage.
By contrast, a third study using the same Dermo1-cre andβ
-catenin conditional mouse lines reported an earlier arrest at a
Runx2+, Osx1- stage
(Hu et al., 2005). Thus,
although there is some disagreement as to the phenotypical outcomes, all
studies indicate that β-catenin activity within skeletal elements is
required for formation of mature osteoblasts. Furthermore, the failure to
complete an osteogenic program was associated with the appearance of ectopic
chondrocytes, suggesting a potential link between canonical Wnt signaling and
inhibition of a chondrogenic pathway within osteoblast progenitors
(Hill et al., 2005;
Day et al., 2005).

As these models remove β-catenin function broadly within the skeletal
anlage, not specifically in the osteoblast lineage, the issue of whether
canonical Wnt/β-catenin activity acts directly within the osteoblast
lineage to promote an osteogenic program remains to be resolved. Here, we have
used a novel Osx1-GFP::Cre mouse strain to investigate the direct roles of
hedgehog and canonical Wnt signaling in early Runx2+,
Osx1+ osteoblast precursors and their derivatives. These data
demonstrate an essential role for canonical Wnt signaling, but not for Hh
signaling, in the progression of osteoblast precursors to mature,
matrix-secreting osteoblasts. Interestingly, cell fate analysis demonstrates
that removal of β-catenin activity in Runx2+,
Osx1+ osteoblast precursors gives rise to ectopic
chondrocytes, suggesting, along with earlier data
(Hill et al., 2005;
Day et al., 2005), an extended
role for canonical Wnt signaling in the suppression of an alternate
chondrocytic fate within osteoblast precursors. In contrast to
loss-of-function studies, enhanced β-catenin activity rapidly accelerates
this program leading to a dramatic expansion of osteoblast precursors and the
premature synthesis of a mineralized bone matrix in the long bones. However,
differentiation to a terminal osteocalcin+
(Oc+; Bglpa1 - Mouse Genome Informatics)
osteoblast is blocked by stabilization of β-catenin. Thus, canonical
Wnt/β-catenin signaling plays crucial roles at specific stages of the
osteogenic program.

Skeletal analysis

In our studies, we failed to observe any significant differences between
skeletons of Osx1-GFP::Cre and β-cateninc/n
embryos and those of wild-type litter mates. Data are shown whereβ
-catenin removal is compared with β-cateninc/n
individuals to control for reduced levels of β-catenin activity where
only one allele is active. Skeletons were stained as described previously
(Long et al., 2001). Embryonic
limbs were dissected and fixed in sodium phosphate-buffered 4%
paraformaldehyde over night at 4°C. Limbs were then washed in
phosphate-buffered saline and either transferred to and stored in 70% ethanol
prior to being paraffin-wax processed, embedded and sectioned, or
cryo-protected in 30% sucrose prior to being frozen mounted in OCT and
sectioned. In situ hybridization with 35S-labeled probes and BrdU
analysis of cell proliferation were carried out as described previously
(Long et al., 2001).
Hematoxylin/eosin, von Kossa and Safranin O staining were performed using
standard histological methods. TUNEL analysis was performed using the ApopTag
in situ Apoptosis Detection Kit (Chemicon), as described in the manufacturer's
instructions.

RESULTS

Removal of canonical Wnt signaling prior to osteoblast
specification

Earlier reports (Hu et al.,
2005; Day et al.,
2005; Hill et al.,
2005) where β-catenin activity was removed prior to or after
cartilage condensation have come to somewhat different conclusions about the
requirement for canonical Wnt/β-catenin signaling in the initial
specification of osteoblast progenitors. In a similar approach, we generated
Col2α1-cre3;β-cateninc/c
conditional knockout embryos in which β-catenin function was specifically
removed from the mesenchyme-derived cartilaginous condensates that give rise
to both the cartilage and the bone of the skeleton
(Long et al., 2001). Hence
Col2α1-cre3 is active both in osteoblasts and in
chondrocyte lineages.

Although the size and structure of the mutant growth plate was comparable
between β-cateninc/c orβ
-cateninc/+ littermates, and vascular invasion and
seeding of the marrow cavity was evident from the presence of red blood cells
within the forming marrow cavity, no histologically identifiable bone matrix
was observed in the mutant long bones (Fig.
1).

These observations were corroborated by molecular analysis.
Col1α1 and Runx2, markers of early osteoblast
progenitor cells, were expressed in mutant tibia at E18.5, albeit at lower
levels than in β-cateninc/c littermates.
Specifically, Col1α1 was expressed by cells within a
wedge-like structure of mesenchyme invading into the presumptive marrow
cavity. Furthermore, Osx1, a marker for osteoblast precursor cells
whose function is downstream of Runx2
(Nakashima et al., 2002), was
expressed in the perichondrial region where osteoblast progenitors first
arise, coincidental with the appearance of
Col1α1-expressing cells in the invading mesenchymal
wedge. By contrast, Oc, a definitive marker gene of terminally
differentiated osteoblasts, was absent from most long bones; the occasional
Oc+ cell observed most likely reflected mosaicism in the
activity of the cre transgene
(Fig. 1; data not shown). These
results are in general agreement with recent studies
(Hu et al., 2005;
Day et al., 2005;
Hill et al., 2005). However,
our observation that, despite robust Osx1 expression, no mineralized
bone matrix is formed suggests that canonical Wnt signaling may be required at
a later stage downstream of Osx1 in the osteogenic program.

Generation of a mouse line expressing a GFP::Cre fusion protein under
the regulation of the Osx1 promoter

Although these experiments are consistent with an intrinsic requirement for
canonical Wnt/β-catenin signaling within the developing osteoblast
lineage, we cannot rule out an alternative role for canonical
Wnt/β-catenin signaling in the cartilage component that might indirectly
regulate the osteoblast pathway. Indeed, cartilage development is perturbed in
this and other similar models in which early growth and hypertrophic
differentiation are disrupted (Hu et al.,
2005; Day et al.,
2005; Hill et al.,
2005) (data not shown), and Wnt signaling is itself implicated in
the chondrogenic program (Day et al.,
2005). Furthermore, hypertrophic chondrocyte-derived factors
(notably Ihh) play a crucial role in endochondral osteoblast development
(reviewed by Kronenberg,
2003). To address signaling directly in the osteoblast lineage, we
generated a BAC transgenic mouse line in which expression of a Tet-off
regulatable GFP::Cre fusion protein is placed under the transcriptional
regulation of the Osx1 promoter. The Tet-off cassette provides an
additional level of potential temporal control of GFP::Cre activity within the
osteoblast lineage that has not been examined in this study. The data herein
and our unpublished data (S.J.R. and A.P.M.) indicate that this is an
effective strategy for the dual regulation of gene activity. However,
unrelated studies in which this regulatory system has been introduced by gene
targeting into several loci suggest that this strategy can lead to dominant
phenotypes. These are not always observed, as in this strategy, suggesting a
gene-, cell type- and/or context-dependent mechanism, the nature of which is
unclear.

Generation of an Osx1-GFP::Cre transgenic mouse line.
(A) Schematic outlining the linear configuration of the pTGCK cassette
used for homologous recombination to the first exon of the osterix1 locus
contained within the BAC: RP23-399N14. The correctly targeted BAC was
subsequently used to generate a transgenic mouse line by pro-nuclear
injection. (B-K) Founder Osx1-GFP::Cre male transgenic mice
were crossed to the female Rosa26lacZ reporter line and activity of
the transgenic line was observed by way of whole-mount lacZ assay at
(B) E14.5, and by lacZ assay or direct fluorescence microscopy on 15μ
m cryosections of tibia from (C,D) E14.5, (F,G) E18.5 and (I,J) postnatal
day 10 mice. (E,H,K) Negative control specimens for littermates, at each
corresponding age, that do not carry the transgene.

As an initial step to characterize expression and activity of the
Osx1-GFP::Cre transgene, transgenic mice were mated to
Rosa26lacZ reporter (R26R) mice
(Soriano, 1999). lacZ
expression, indicative of GFP::Cre activity, was observed in all boney
elements (endochondral and membranous) consistent with endogenous
Osx1 expression (Nakashima et
al., 2002) (Fig.
2B). Bone-specific activity was evident when GFP::Cre fluorescence
and lacZ expression were examined in tibial sections
(Fig. 2C,D). GFP::Cre and
lacZ expression were observed in the inner bone-forming
perichondrium, adjacent to hypertrophic chondrocytes, and sporadically in
hypertrophic chondrocytes. Similarly, analysis at E18.5
(Fig. 2F-H) and postnatal day
10 (Fig. 2I-K) demonstrated
that expression of lacZ and GFP::Cre fluorescence were largely
restricted to the periosteum and primary spongiosa. Thus,
Osx1-GFP::Cre is active specifically within the osteoblast lineage
and expression is maintained throughout embryonic and early postnatal
development. The fact that GFP::Cre activity is largely absent from
chondrocytes in which Osx1 normally shows weak expression
(Nakashima et al., 2002)
suggests either that low-level GFP::Cre activity is inefficient in initiating
recombination, or, alternatively, that chondrocyte-specific regulatory
elements are absent in the BAC transgene.

An intrinsic requirement for β-catenin in the progression of
Osx1+ cells to bone-producing osteoblasts

To determine whether β-catenin is required downstream of Osx1
in the osteoblast lineage,
Osx1-GFP::Cre;β-cateninc/n conditional
knockout embryos were collected at E14.5, E16.5 and E18.5, and their gross
skeletal organization analyzed (Fig.
3). At E14.5,
Osx1-GFP::Cre;β-cateninc/n mutant skeletons
were comparable in size to wild-type embryos. At E16.5, no Alizarin red
staining was observed despite clear zones of hypertrophy in mutant limbs. By
E18.5, mineralization of the endochondral skeletal elements was visible;
however, as in the Col2α1-cre3 removals, the remaining
mineralization was associated with hypertrophic chondrocytes. Importantly, in
contrast to the Col2α1-cre3 removal of β-catenin
activity, which is specific to the endochondral skeleton,
Osx1-GFP::Cre;β-cateninc/n embryos lacked
the membranous bone of cranial ossification centers. Hence, there is a
complete loss of bone deposition that is reminiscent of the loss of osteoblast
determinants, such as Runx2
(Komori et al., 1997;
Ducy et al., 1997;
Otto et al., 1997) and
Osx1 (Nakashima et al.,
2002).

Histological analysis of long bones at E14.5 showed that the size and
organization of the developing tibia was comparable to wild type with respect
to chondrocyte differentiation and proliferation, as we had expected from the
demonstrated specificity of the Osx1-GFP::Cre transgene (data not
shown). Analysis of tibial elements at E16.5 and E18.5 indicated that the size
and structure of the growth plate was comparable to wild type. The only
detectable mineralized matrix was associated with hypertrophic chondrocytes
and, in addition to the normal growth plate, hypertrophic chondrocytes
ectopically lined the periosteal region
(Fig. 4 and data not shown).
Vascular invasion is critically linked with osteogenesis; however, the
adjacent forming marrow cavity was well vascularized, and Mmp9, Mmp13
and Vegf, which are associated with vascular invasion were expressed
normally (Fig. 4). Together,
these observations support a primary role for β-catenin downstream of
Osx1 in osteoblast specification.

To characterize osteoblast development, we examined expression of the
osteoblast cell state marker genes Col1α1, Runx2, Osx1
and Oc (Fig. 4).
Expression of Col1α1, Runx2 and Osx1 was
observed in the periosteal region and in a mesenchymal wedge invading the
marrow cavity in E18.5
Osx1-GFP::Cre;β-cateninc/n mutant tibia
(Fig. 4). Thus, as expected
Osx1+ osteoblasts were present. However, no
Oc+ osteoblasts were detected indicating a failure of
osteoblast progression to terminal Oc+ osteoblasts in
mutant embryos. No apparent difference was observed in either cell death or
cell proliferation between wild-type and mutant tibia (data not shown).

A transient requirement for Ihh upstream of canonical
Wnt/β-catenin signaling in osteoblast development

Several signaling systems are known to play important roles during
osteoblast development (reviewed by
Kronenberg, 2003;
Karsenty, 2003). Ihh
null mice fail to differentiate osteoblasts, but arrest in this pathway
appears to occur at an initiating step: the transition of an unspecified cell
to a pre-osteoblast (St-Jacques et al.,
1999; Long et al.,
2004). As expected, the Ihh expression domain of
prehypertrophic chondrocytes was present, consistent withβ
-cateninc/n embryos, and active Ihh signaling was
evident from the upregulation of Ptch1 (the Hh receptor and primary
target of Hh signaling) in the periosteal region where osteoblast
specification initiates (Fig.
4).

Further insight into the relationship between Hh and Wnt function comes
from the osteoblast-specific removal of Hh signaling in
Osx1+ osteoblast precursors and their descendents. As
discussed earlier, Ihh is required for the specification of early
Runx2+ osteoprogenitors, prior to the requirement for
canonical Wnt signaling. However, whether this represents a transient signal
input or the onset of a more extended role for Ihh signaling in osteoblast
development has not been addressed. We generated
Osx1-GFP::Cre;Smoc/n embryos to remove smoothened (Smo)
activity (and, consequently, all responsiveness to Hh signaling) after
specification of a Runx2+;Osx1+ osteoblast
progenitor. Analysis of Osx1-GFP::Cre;Smoc/n embryos at
E18.5 by whole-mount skeletal preparation, histological analysis and in situ
hybridization with chondrogenic and osteogenic marker genes demonstrated a
normal program of chondrocyte and osteoblast development
(Fig. 5; data not shown). Thus,
Hh signaling does not appear to play an essential role in the terminal
differentiation of osteoblasts beyond an Osx1+ cell
state.

Osteoblast to chondrocyte cell fate changes in the absence ofβ
-catenin activity in the osteoblast lineage

Previous studies have indicated that when osteoblast differentiation is
arrested prior to Osx1 expression the loss of a terminally
differentiated osteoblast is accompanied by the appearance of ectopic
chondrocytes (Nakashima et al.,
2002; Long et al.,
2004; Hill et al.,
2005; Day et al.,
2005). Consistent with this view, analysis of the expression of
collagen 2α1 (Col2α1) and collagen 10α1
(Col10α1), markers of proliferating and postmitotic
hypertrophic chondrocytes, highlight ectopic chondrocytes lining the
periosteal region and an invading mesenchymal wedge adjacent to
Col1α1 (a marker of early osteoblast progenitors),
Runx2 and Osx1 expressing cells (Figs
1,
4). Thus, the presence of
Runx2 and Osx1 within an osteoblast precursor does not
appear to be sufficient to maintain an osteogenic program in the absence ofβ
-catenin; osteoblast precursors convert to a chondrocyte fate uponβ
-catenin removal. This conclusion was further supported by performing
double-labeled fluorescent in situ hybridization on wild-type and mutant
tibia, comparing the expression of Col2α1 and
Col10α1 with the activity of the
Osx1-GFP::Cre transgene. Furthermore, Osx1-GFP::Cre-mediated
activation of the R26R allele in
Osx1-GFP::Cre;β-cateninc/n;R26R mice
demonstrates that the ectopic chondrocytes arise from
Osx1-GFP::Cre-expressing osteoblast precursors (data not shown).

Stabilization of β-catenin in osteoblast precursor cells results
in premature mineralization and increased proliferation of
pre-osteoblasts

Osx1-GFP::Cre;Catnblox(ex3)/+ mutants died at birth.
Skeletal preparations at E14.5, E16.5 and P0 revealed that mutant embryos,
overall, had shorter limbs in comparison with
Catnblox(ex3)/+ wild-type littermates
(Fig. 6). Alizarin red staining
of the mineralized bone matrix was first evident at E16.5 in whole-mount
skeletons. At this time, mutants appeared to have an intense and broader
ossification center in the long bones in comparison with their wild-type
counterparts, although ossification in the skull bones was delayed. By P0, a
thick bony matrix characterized all long bones in the mutants, and bone
formation was now visible in several cranial regions.

Histological analysis of the long bones at E14.5 and E16.5 showed, an
abnormal wedge-shaped growth plate with very few identifiable hypertrophic
chondrocytes when compared with wild-type tibias (Figs
7,
8). Analysis of histological
sections at E13.5 (data not shown) and E14.5
(Fig. 7) using von Kossa and
Alizarin red staining techniques revealed extensive premature mineralization
of the mutant tibia at this time; no mineralization was detected in the wild
type, even at E14.5 (Fig. 7).
At E16.5, the primary spongiosa was readily identifiable in the wild-type
tibia but no primary spongiosa-like matrix was observed in the mutant. Rather,
the matrix resembled the dense matrix normally restricted to the cortical
bone-forming region that generates the bone collar
(Fig. 8).

To characterize the observed premature ossification of the mutants, we
examined expression of the osteoblast differentiation markers
Col1α1, Osx1 and Oc. While
Col1α1 was expressed in the periosteal region of the
wild type at E14.5, and the primary spongiosa at E16.5, expression of
Col1α1 was observed throughout the periosteal region
and entire central region of the tibia in the mutants
(Fig. 7; data not shown). A
similar dramatic expansion was also observed in the expression of
Osx1, suggesting that the stabilization of β-catenin in
osteoblast precursor cells resulted in the promotion of osteoblast
development. Although the synthesis of bone matrix was activated prematurely
at E13.5, we failed to observe Oc+ terminal osteoblasts
prior to the normal onset of Oc expression at E16.5 (data not shown).
Furthermore, although Oc+ osteoblasts were clearly
identifiable in the wild-type tibia at E16.5, Oc+
osteoblasts were rare in the mutant; when present, these cells were
exclusively restricted to the periosteal region and expressed low levels of
Oc relative to their wild-type counterparts
(Fig. 8). The expansion of the
osteoblast lineage was accompanied by a 3-fold increase in proliferation in
osteoblast-forming regions along the length of the periosteum in mutants at
E14.5 (Fig. 7). Together, these
observations suggest that the stabilization of β-catenin results in a
marked increase in proliferation of an Osx1+ precursor
population and an accelerated progression of an osteoblast program to mature
bone-secreting osteoblasts. Although these cells actively synthesize a bone
matrix, they do not progress to a terminal state, characterized by high
Oc+ expression. Thus, the cessation of canonical
Wnt/β-catenin signaling may normally accompany this progression in vivo.
As most Oc+ cells are normally observed in the marrow
cavity, Oc+ expression may reflect an absence of local
canonical Wnt signaling in this region.

Recent evidence indicates that canonical Wnt/β-catenin signaling
positively regulates bone matrix formation by suppressing osteoclast
development (Glass et al.,
2005). Thus, a failure of osteoclast development, and,
consequently, a loss of bone matrix turnover, could explain the premature
formation of a bone matrix following osteoblast-specific stabilization ofβ
-catenin. However, when wild-type limbs were examined, it was evident
that Trap-positive osteoclasts were not present at E14.5 (data not
shown). Thus, the accelerated bone matrix phenotype precedes any role for
osteoclasts in bone remodeling. However, when osteoclasts were normally
detected in the wild type at E16.5, they were completely absent from the
mutant (data not shown). Consistent with the findings of Glass et al.
(Glass et al., 2005), that the
osteoclast inhibitor osteoprotegerin (Opg; Tnfrsf11b - Mouse
Genome Informatics) is regulated by canonical Wnt signaling, the failure of
osteoclast formation correlated with a dramatic upregulation of Opg
upon osteoblast-specific stabilization of β-catenin relative to wild-type
osteoblasts at E14.5 (Fig. 7;
data not shown).

Osx1-GFP::Cre conditional stabilization of β-catenin does
not promote expression of osteocalcin. Histological analysis and in situ
hybridization of an osteocalcin-specific anti-sense 35S-labeled
riboprobe to sections of E16.5 tibia from (A-E)
Catnblox(ex3)/+ (wild type) and (F-J)
Osx1-GFP::Cre;Catnblox(ex3)/+ (mutant) embryos. Arrows
indicate sites of high osteocalcin expression in the wild type and low-level
osteocalcin expression in the mutant in the periosteum.

Analysis of Dkk1 and Tcf1 expression (Wnt pathway
modulators and targets) indicated that active Wnt signaling was clearly
upregulated in the osteoblast population in response to the activation ofβ
-catenin (data not shown), which is consistent with the activation of
canonical Wnt signaling and direct regulation of Dkk1 and
Tcf1 transcription by β-catenin. Despite over-mineralization of
the tibia, vascular invasion was evident, and mutants expressed Mmp9
and Mmp13 (data not shown). In addition, the population of
Col10α1- expressing hypertrophic chondrocytes was
clearly reduced (Fig. 7; data
not shown). TUNEL analysis revealed that mutants displayed a significant
increase in cell death at the interface of hypertrophic chondrocytes and the
mineralized matrix, suggesting that TUNEL-positive cells were likely to
represent hypertrophic chondrocytes (data not shown). Finally, we examined the
expression of indicators of Ihh signaling. These appeared to be largely
unaltered, suggesting that the observed increase in bone matrix does not
result from an overt change in the Hh-regulation of osteoblast precursors
(data not shown).

Wnt7b is not essential for the terminal differentiation of
osteoblasts

The nature of the putative Wnt ligand that would mediate Wnt action in the
osteoblast lineage is unclear. A recent report has highlighted Wnt7b
as a candidate (Hu et al.,
2005). Wnt7b expression was reported in the
osteoblast-forming region of the long bone; furthermore, expression was
dependent upon Ihh signaling, and was upregulated in response to Hh signaling
in Hh-stimulated osteoblast specification of C3H10T1/2 mesenchymal progenitor
cells (Hu et al., 2005).
However, we recently generated embryos in which all Wnt7b function
was removed and we have not observed any defects in skeletal development
(S.J.R. and A.P.M., unpublished).

DISCUSSION

We have used genetic approaches to modify Hh and Wnt signaling in the
developing osteoblast lineage of the mouse. These data point to sequential and
genetically separable roles for these signaling pathways in the progressive
specification of osteoblasts within the endochondral skeleton
(Fig. 9).

Osteoblast progenitors can first be identified within the inner
perichondrium adjacent to, and coincident with, the first appearance of
hypertrophic chondrocytes. This tight linkage reflects a crucial role for Ihh
signaling (St-Jacques et al.,
1999; Chung et al.,
2001). Ihh is produced by pre-hypertrophic chondrocytes and
appears to act directly on perichondrially located osteoblast progenitors to
specify the osteoblast precursors
(St-Jacques et al., 1999;
Long et al., 2004). To date,
all markers of this program in the endochondral-, but not the membranous
bone-, derived skeleton are dependent on an initial Ihh input. Furthermore, Hh
activates osteoblast development in a variety of mesenchymal and skeletogenic
cell types in vitro (Nakamura et al.,
1997; van der Horst et al.,
2003; Long et al.,
2004). In the absence of signaling, perichondrial osteoblast
progenitors in the perichondrium adopt a chondrocyte fate, as evidenced by the
layer of immature chondrocytes that surround the hypertrophic chondrocytes in
Ihh mutants (St-Jacques et al.,
1999), and by the ectopic chondrogenesis exhibited by
perichondrially localized Smo mutant cells in chimeric mice
(Long et al., 2004). Although
this suggests that a potential cell, frequently termed an
osteochondroprogenitor (OCP), resides within the perichondrium, a conclusion
supported by clonal analysis in vitro
(Nakase et al., 1993), there
is currently no evidence that these cells ordinarily give rise to
chondrocytes. Thus, during normal development, Ihh signaling appears to act as
a switch within a specific population of inner perichondrial mesenchyme to
initiate a program of bone formation. Failure to activate this switch results
in cells adopting an alternative chondrocyte pathway of development
(Fig. 9). Given the crucial
role for Ihh signaling in regulating the temporal and spatial program
of early osteoblast commitment, what role does Ihh play beyond this
stage? Our results do not support an on-going role for Ihh signaling in
progression along the osteoblast pathway of differentiation
(Fig. 5). When Smo
activity is removed in Osx1+ osteoblast precursors, normal
bone secreting OcHigh osteoblasts are generated, and the
endochondral skeleton at birth is indistinguishable from wild type. Whether
this is also true in the adult is currently under investigation. Clearly,
Ihh continues to be expressed postnatally within prehypertrophic
chondrocytes, and the upregulation of Ihh signaling at sites of
fracture repair implicates Ihh in skeletal homeostasis, repair and
regeneration (Vortkamp et al.,
1998; van der Eerden et al.,
2000; Le et al.,
2001).

Proposed model for the role of Hh and canonical Wnt signaling in
regulating the differentiation of skeletal progenitors. Various lines of
evidence implicate the importance of canonical Wnt signaling at multiple
stages along the osteoblast differentiation pathway, from the specification of
early skeletal progenitor cells to a terminally differentiated osteoblast.
Observations from these studies (Hu et
al., 2005; Day et al.,
2005), together with results presented in this study, have been
used to synthesize a working model for the specific functions of Hh and
canonical Wnt signaling during osteoblast specification and differentiation.
See text for details. Red and green arrows indicate the requirement for Hh and
canonical Wnt signaling, respectively; the blue line represents the negative
regulation of osteoclasts by OPG.

In contrast to Ihh, the manipulation of β-catenin activity provides
evidence against an essential role for canonical Wnt signaling in early
osteoblast specification. When β-catenin activity was removed in skeletal
progenitors with Prx1-cre, Runx2+ prior to specification
of the skeletal anlage in the limbs, osteoblast precursors were specified, but
these failed to progress to Osx1+ precursors and no bone
matrix was laid down (Hill et al.,
2005) (Fig. 9).
Although the precise kinetics of the inactivation of the β-catenin
conditional allele and turnover of existing β-catenin protein in this
model are not clear, it seems reasonable to conclude from the early expression
of the Cre driver that β-catenin is not essential for the specification
of a Runx2+ osteoblast precursor. Furthermore, the results
are consistent with a role for canonical Wnt/β-catenin signaling in the
transition to an Osx1+ osteoblast precursor. Other studies
using later active Cre driver mice that, like Prx1-cre, are active in
both the chondrocyte and osteoblast compartments of the endochondral skeleton
give conflicting results. Although all reports fail to observe a role forβ
-catenin in the initial specification of Runx2+
osteoblast precursors, using an identical Dermo1-cre transgenic line
to remove β-catenin from pre-cartilaginous endochondral skeletal
condensates, Hu et al. (Hu et al.,
2005) observed an arrest of osteoblast specification at a
Runx2+, Osx1- stage, whereas Day et al.
(Day et al., 2005) reported a
progression, albeit at reduced levels, to a Runx2+,
Osx1+ precursor and arrest at this stage, suggesting a
continuing role for β-catenin in osteoblast differentiation beyond the
Osx1+ osteoblast precursor.

This interpretation is also supported by
Col2α1-cre-mediated removal
(Day et al., 2005) (this study;
Fig. 9). However, the
occasional appearance of OcHigh osteoblasts
(Day et al., 2005) (this study)
and the broad activity of these Cre strains within both osteoblast and
chondrocyte lineages prevents a rigorous assessment of the specific direct
roles for canonical Wnt/β-catenin signaling in osteoblast
differentiation.

Our data, in which β-catenin activity is removed specifically from
Runx2+, Osx1+ osteoblasts, provides compelling
evidence that β-catenin is essential within the osteoblast lineage for
the specification of an Osx1+ osteoblast to a
bone-secreting osteoblast (Fig.
9). Furthermore, the demonstration that the stabilization ofβ
-catenin, and, consequently, the activation of canonical Wnt signaling,
within osteoblast precursors expands this population and accelerates the
progression to a bone matrix-secreting osteoblast indicates that canonical
Wnt/β-catenin signaling may regulate both the proliferation and
maturation of the osteoblast precursor pool. Interestingly, continued
activation of canonical Wnt/β-catenin signaling arrests osteoblasts at an
OcLow stage, suggesting that progression to an
OcHigh state may require the downregulation of this
signaling input in the primary spongiosa where the majority of
OcHigh cells are located
(Fig. 9).

In addition to a proposed cell autonomous role for β-catenin in
promoting a bone-secreting osteoblast pathway, recent studies suggest that
canonical Wnt/β-catenin signaling may play a non-cell autonomous role in
suppressing the development of bone-matrix-degrading osteoclasts through the
production of OPG (Glass et al.,
2005). Our data lend further support to this view. We observed a
dramatic upregulation of Opg upon stabilization of β-catenin in
Osx1+ osteoblast precursors. However, as the normal
appearance of osteoclasts occurs after the onset of bone matrix secretion in
this model, the premature bone matrix deposition observed at E14.5 precedes
any role for osteoclast suppression in this early phenotype. By contrast,
OPG-mediated inhibition of osteoclast development may contribute to the
massive expansion of a cortical bone-like matrix in E16.5 long bones of
Osx1-GFP::Cre;Catnblox(ex3)/+ embryos.

Interestingly, in earlier reports, in which Cre removed β-catenin
broadly in skeletal structures, ectopic chondrocytes were observed in
perichondrial regions where cortical bone first arises
(Hill et al., 2005;
Day et al., 2005). Here, we
show that the osteoblast-specific removal of β-catenin results in
osteoblast precursors in which Cre was active, adopting a chondrocyte-like
fate. These ectopic chondrocytes appear to undergo a transition from early
Col2α1 immature chondrocytes to
Col10α1 hypertrophic chondrocytes, as in the normal
growth zone. Thus, β-catenin acts both to promote an osteoblast program
and to block an alternative program of chondrogenesis within osteoblast
precursors: an Ihh-mediated activity at an earlier stage in the osteoblast
lineage.

How do Hh and Wnt signals act at the molecular level to sequentially
regulate osteoblast differentiation? Hh signaling has been shown to modify
cellular responsiveness to other signals, most notably several members of the
Bmp family (Murtaugh et al.,
1999). Bmps are implicated in the specification of both
chondrocytes and osteoblasts (reviewed by
Hoffmann and Gross, 2001), as
well as in the subsequent modification of the osteogenic program, where some
Bmps promote bone formation [such as Bmp2, Bmp7, Bmp6 and Bmp9
(Peng et al., 2003)], although
Bmp3 acts as a negative regulator of bone formation
(Daluiski et al., 2001). In
vivo, Bmp signaling is essential for the normal induction of endochondral
anlage, and, both in vivo and in vitro, Bmps can promote an osteoblast program
of mesenchymal cell differentiation. One possible role for Ihh signaling is to
alter the responsiveness of perichondrial osteoblast progenitors to a Bmp
input, modifying the response from chondrocyte inducing to osteoblast
inducing. Consistent with this model, Bmp-mediated induction of an osteoblast
phenotype in vitro requires prior Ihh signaling
(Long et al., 2004), and has
been shown to function, at least in part, by induction of a Wnt autocrine loop
(Rawadi et al., 2003). In
addition, Bmp2 signaling has been shown to upregulate the expression of
Sox9 (Healy et al.,
1999; Zehentner et al.,
1999), a key early determinate of chondrocytes, whereas
Sox9 is downregulated in the perichondrial region where osteoblast
differentiation initiates (Yamashiro et
al., 2004). Whether Sox9 downregulation is Ihh dependent
is unclear but, given the ectopic chondrocyte phenotype adopted by
perichondrially localized Smo mutant cells, it is reasonable to
presume that this, as with normal chondrogenesis, requires high levels of
Sox9 activity.

The transcriptional activation domain of Sox9 has also been reported to
directly interact with specific armadillo repeats in β-catenin. On the
basis of these interactions, it has been proposed that Sox9 inhibitsβ
-catenin activity, promoting β-catenin degradation
(Akiyama et al., 2004), such
that overexpression of Sox9 generates a similar phenotype to
loss-of-function of β-catenin. Thus, the balance of Sox9 andβ
-catenin may regulate alternate programs of chondrocyte and osteoblast
development, respectively. When Sox9 levels are high and β-catenin levels
are low, a chondrocyte program may be favored. By contrast, high levels ofβ
-catenin and low levels of Sox9 may act on appropriately specified
progenitors to promote an osteoblast fate. Interestingly,
Prx1-cre-mediated stabilization of β-catenin in skeletal
progenitors leads to a dramatic loss of the endochondral skeleton, suggesting
that the timing of action of β-catenin (downstream of Ihh in
perichondrial osteoblast precursors) may be crucial to its normal osteoblast
role (Hill et al., 2005).

Canonical Wnt ligands have been shown to stimulate Runx2
expression and Runx2 is itself essential for osteoblast development
(Gaur et al., 2005). However,
in our study, and in other studies discussed earlier, β-catenin is not
essential for the initial activation of Runx2. These findings do not exclude
the possibility that the Wnt input may normally regulate either the level or
duration of Runx2 expression in the osteoblast lineage. By contrast,
Prx1-cre removal of β-catenin suggests that Osx1
expression is β-catenin dependent. Analysis of the expression of each
gene in osteoblast precursors in Runx2 and Osx1 mutants
indicates that Osx1 activation lies downstream of Runx2
(Nakashima et al., 2002); thus
Runx2, although required in vivo for Osx1 activation, is not
sufficient in the absence of β-catenin. Furthermore, Bmp2 activates
Osx1 in Runx2 mutant cells
(Lee et al., 2003); thus, the
molecular hierarchies and interactions underlying Osx1 activation are
uncertain, although its crucial role in the specification of all osteoblasts
has been clearly demonstrated (Nakashima
et al., 2002).

After the initial appearance of a bone matrix, a subset of osteoblasts
activates Oc; Oc is considered a late marker of terminal osteoblasts.
Oc is itself a direct target of Runx2 regulation, where Runx2 binds a
cis-regulatory region within the Oc promoter
(Paredes et al., 2004). Our
data indicate that when β-catenin levels remain high in the osteoblast,
secretion of a bone matrix is promoted, but osteoblasts express only low
levels of Oc (OcLow,
Fig. 9). Thus, a loss of a
canonical Wnt input appears to accompany the progression to an
OcHigh state. Interestingly, Lef1, aβ
-catenin-binding partner and target of canonical Wnt/β-catenin
signaling in the osteoblast lineage (Hu et
al., 2005), has been shown to inhibit Runx2-mediated activation of
the Oc promoter; Lef1- and Runx2-binding sites lie adjacent to each
other in the relevant cis-regulatory region within Oc
(Kahler and Westendorf, 2003).
Thus, the direct integration of distinct regulatory inputs on the Oc
promoter may explain the observed block in Oc activation in our
study.

Finally, what is the identity of the postulated Wnt signal(s) mediating the
proposed canonical Wnt signaling? Several Wnts have been reported to be
expressed in the developing skeletal anlage
(Hu et al., 2005). Of
particular interest is Wnt7b. Wnt7b is expressed in the bone-forming
region downstream of Ihh, providing a potential link between the Ihh
and Wnt pathways (Hu et al.,
2005). However, although Wnt7b may play a redundant role with
another factor, our data indicate that Wnt7b is not essential for
osteoblast development. Thus, as with other regions of the embryo where
studies of β-catenin action have made a strong case for a canonical Wnt
input, most notably in stem cell maintenance in the mammalian gut (reviewed by
Pinto and Clevers, 2005), the
regulatory ligand(s) remains to be identified.

Acknowledgments

We would like to thank Dr M. Todd Valerius for providing the pTGCK vector,
Drs Tom Carroll, Jing Yu and Jay Rajagopal for Wnt7b skeletal
analysis, and Dr Ernestina Schipani for providing in situ probes and advice
and support for histological analysis. S.J.R. was supported by fellowships
from the National Health and Medical Research Council of Australia (#301299)
and the Arthritis Foundation (#401683). Work in A.P.M.'s laboratory was
supported by a program grant from the NIH (NIH-NIDDK PO1 #DK56246).

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